This invention relates to novel compositions comprising nanocrystals which are both functionalized and have enhanced fluorescent properties. More particularly, the present invention relates to coating nanocrystals with a compound which imparts to the coated nanocrystals properties which include water-solubility, functionalization, and an unexpected enhancement of fluorescence intensity.
Fluorescence-based analyses and nonisotopic detection systems have become a powerful tool and preferred mode in scientific research and clinical diagnostics, as well as in many industrial applications, for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, immunoassays, histochemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluorescein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, photobleaching (fading of intensity under light sources) is a major problem that hinders the accuracy of quantitative measurements using these molecules. In addition, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule due to the relatively narrow absorption spectrum of each. Even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), there is often insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap. That is, typical fluorescent dyes each have an emission spectrum that is rather broad which often limits combinations of fluorescent molecules that can be used simultaneously. Additionally, conventional fluorescent dyes have limited fluorescence intensity over time (suffer from photobleaching). Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules that can be used to label a target molecule to be detected.
Semiconductor nanocrystals are now being evaluated for their adaptation to detection systems in aqueous environments. An advantage of such nanocrystals is that they can be produced in a narrow size distribution and, since the spectral characteristics are a function of the size, can be excited to emit a discrete fluorescence peak of narrow bandwidth. In other words, the ability to control the spectral characteristics of nanocrystals (narrow bandwidth, discrete emission wavelengths, a single wavelength can excite an array of nanocrystals with different emissions) is the major attracting point in their use. Another advantage of the nanocrystals is their resistance toward photobleaching under intensive light sources. As known in the art, a manual batch method may be used to prepare semiconductor nanocrystals of relative monodispersity (e.g., the diameter of the core varying approximately 10% between quantum dots in the preparation), as has been described previously (Bawendi et al., 1993, J. Am. Chem. Soc. 115:8706). Advances in nanocrystal core production and reductions in average particle size have been achieved by a continuous flow process (U.S. Pat. No. 6,179,912, the disclosure of which is herein incorporated by reference). Additionally, semiconductor nanocrystals of different sizes may be excited with a single spectral wavelength of light.
Examples of semiconductor nanocrystals are known in the art to have a core selected from the group consisting of CdSe, CdS, CdTe (collectively referred to as “CdX”)(see, e.g., Norris et al., 1996, Physical Review B. 53:16338-16346; Nirmal et al., 1996, Nature 383:802-804; the disclosures of which are hereby incorporated by reference).
Core semiconductor nanocrystals, however, exhibit low fluorescence intensity upon excitation, and additionally, lack water-solubility. The low fluorescence intensity has been ascribed to the presence of surface energy states that act as traps which degrade the fluorescence properties of the core nanocrystal.
Efforts to improve the fluorescence intensity involve passivating (or capping) the outer surface of a core nanocrystal in thereby reducing or eliminating the surface energy states associated therewith. Organic molecules, such as tri-n-octyl phosphine (TOP) and tri-n-octyl phosphine oxide (TOPO) have been used for passivation. Inorganic materials have also been used for passivation; i.e., core nanocrystals have been passivated with an inorganic coating (“shell”) uniformly deposited thereon. The shell which is typically used to passivate CdX core nanocrystals is preferably comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se, or even Te. Semiconductor nanocrystals having a CdX core and a YZ shell have been described in the art (see, e.g., Danek et al., 1996, Chem. Mater. 8:173-179; Dabbousi et al., 1997, J. Phys. Chem. B 101:9463; Rodriguez-Viejo et al., 1997, Appl. Phys. Lett. 70:2132-2134; Peng et al., 1997, J. Am. Chem. Soc. 119:7019-7029; the disclosures of which are hereby incorporated by reference). However, the above described passivated semiconductor nanocrystals have been reported to have limited improvements in fluorescence intensity (with reference to quantum yield), and to have solubility in organic, non-polar (or weakly polar) solvents only.
To make fluorescent nanocrystals useful in biological applications or detection systems utilizing an aqueous environment, it is desirable that the fluorescent nanocrystals used in the detection system are water-soluble. “Water-soluble” is used herein to mean sufficiently soluble or suspendable in an aqueous-based solution, such as in water or water-based solutions or buffer solutions, including those used in biological or molecular detection systems as known by those skilled in the art. One method to impart water-solubility to semiconductor nanocrystals (e.g., CdX core/YZ shell nanocrystals) is to exchange the overcoating layer of TOP or TOPO with a coating, or “capping compound”, which will impart some water-solubility. For example, a mercaptocarboxylic acid may be used as a capping compound to exchange with the organic layer (see, e.g., U.S. Pat. No. 6,114,038, the disclosure of which is herein incorporated by reference; see also, Chan and Nie, 1998, Science 281: 2016-2018). The thiol group of monothiol capping compound bonds with the Cd-S or Zn-S bonds (depending on the composition of the nanocrystal), creating a coating which is not easily displaced in solution, and imparting some stability to the nanocrystals in suspension. Further advances in water solubility, stability, and fluorescence properties have been achieved by using novel amino acid coating technology. In preferred embodiments, a diaminocarboxylic acid is either used to exchange with the capping compound, or is used to overlay the capping compound in operative connection therewith (see, e.g., U.S. Pat. No. 6,114,038). Successive amino acid layers may then be added.
Another method to make the CdX core/YZ shell nanocrystals water-soluble is by the formation of a coating of silica around the semiconductor nanocrystals (Bruchez, Jr. et al., 1998, Science 281:2013-2015; U.S. Pat. No. 5,990,479) utilizing a mercapto-based linker to link the glass to the semiconductor nanocrystals. An extensively polymerized polysilane shell has been reported to impart water solubility to nanocrystalline materials, as well as allowing further chemical modifications of the silica surface. However, depending on the nature of the coating compound, coated semiconductor nanocrystals that have been reported as water-soluble may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. For example, oxygen and light can cause mercapto-based monothiols used in coating or linking to become oxidized, thereby forming disulfides which destabilize the attachment of the coating or linking molecules to the nanocrystal. Thus, oxidation may cause the coating or linking molecules to migrate away from the outer surface of the nanocrystals, thereby exposing the surface of the nanocrystals resulting in “destabilized nanocrystals” Destabilized nanocrystals form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the nanocrystals. Additionally, current means for passivating semiconductor nanocrystals are still rather inefficient in increasing the fluorescence intensity to a level desired for detection systems (e.g., in providing a significant increase in sensitivity in fluorescence-based detection systems as compared to currently available fluorescent dyes).
Thus, there remains a need for a nanocrystal that overcomes the above-referenced limitations, and others.